This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project is following a former lab member Derek McLachlin[unreadable]s collaboration with Yong Kim from Dr. Greengard's lab. p35 is a cyclin-dependent kinase 5 (Cdk5) activator. Cdk5/p35 complex phosphorylates diverse substrates which have multifunctional roles in the nervous system. During development, it participates in neuronal differentiation, migration, axon outgrowth and synaptogenesis. P35 was found autophosphorylated in vitro, which may indicate a mechanism of functional regulation in vivo. We set out to identify the p35 phosphorylation sites as an initial step towards studying its function. P35 were over-expressed and purified from insect cells and phosphorylated with cdk5 and 32P-ATP in vitro. Protein was tryptic digested and peptides was separated by HPLC, phosphopeptide were identified by MALDI-TOF followed by tandem MS/MS, as well as HMMS (hypothesis-driven multi-stage MS). Six phosphopeptides were identified which correspondent to two phosphorylation sites. Site directed mutagenesis and 2D peptide map of the tryptic digest of P35 were used to confirm the identified sites. We are currently focusing on identifying an additional phosphorylation site shown by the phospho-image of the 2D map from tryptic digest of phosphorylated wild type p35.